Discovery and Kinetic Profiling of 7-Aryl-1,2,4-triazolo[4,3-a]pyridines: Positive Allosteric Modulators of the Metabotropic Glutamate Receptor 2

Article abstract of DOI:10.1021/acs.jmedchem.7b00669

We report the synthesis and biological evaluation of a series of 7-aryl-1,2,4-triazolo[4,3-a]pyridines with mGlu₂ positive allosteric modulator (PAM) activity and affinity. Besides traditional in vitro parameters of potency and affinity, kinetic parameters k_on, k_off and residence time (RT) were determined. The PAMs showed various kinetic profiles; k_on values ranged over 2 orders of magnitude, whereas RT values were within a 10-fold range. Association rate constant k_on was linearly correlated to affinity. Evaluation of a short, medium, and long RT compound in a label-free assay indicated a correlation between RT and functional effect. The effects of long RT compound 9 on sleep-wake states indicated long RT was translated into sustained inhibition of rapid eye movement (REM) in vivo. These results show that affinity-only driven selection would have resulted in mGlu₂ PAMs with high values for k_on but not necessarily optimized RT, which is key to predicting optimal efficacy in vivo.

Full text of DOI:10.1021/acs.jmedchem.7b00669

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Article
Discovery and kinetic profiling of 7-aryl-1,2,4-triazolo[4,3-a]pyridines:
positive allosteric modulators of the metabotropic glutamate receptor 2.
Maarten L. J. Doornbos, José María Cid, Jordi Haubrich, Alexandro Nunes, Jasper W. van de
Sande, Sophie C. Vermond, Thea Mulder-Krieger, Andrés A. Trabanco, Abdellah Ahnaou, Wilhelmus
H. Drinkenburg, Hilde Lavreysen, Laura H. Heitman, Adriaan P. IJzerman, and Gary Tresadern
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00669 • Publication Date (Web): 13 Jul 2017
Downloaded from http://pubs.acs.org on July 13, 2017
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Discovery and kinetic profiling of 7ꢀarylꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridines: positive allosteric
modulators of the metabotropic glutamate receptor
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Maarten L. J. Doornbos , José María Cid , Jordi Haubrich , Alexandro Nunes , Jasper W. van
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de Sande , Sophie C. Vermond , Thea MulderꢀKrieger , Andrés A. Trabanco , Abdellah
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Ahnaou , Wilhelmus H Drinkenburg , Hilde Lavreysen , Laura H. Heitman , Adriaan P.
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IJzerman , Gary Tresadern .
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Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR),
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Leiden University, P.O. Box 9502, 2300RA Leiden, The Netherlands; Janssen Research and
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Development, Calle Jarama 75A, 45007, Toledo, Spain; Janssen Research and Development,
Turnhoutseweg 30, 2340 Beerse, Belgium.
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ABSTRACT.
We report the synthesis and biological evaluation of a series of 7ꢀarylꢀ1,2,4ꢀtriazolo[4,3ꢀ
a]pyridines with mGlu positive allosteric modulator (PAM) activity and affinity. Besides
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traditional in vitro parameters of potency and affinity, kinetic parameters k , koff and residence
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time (RT) were determined. The PAMs showed various kinetic profiles; k values ranged over
on
two orders of magnitude, whereas RT values were within a 10ꢀfold range. Association rate
constant kon was linearly correlated to affinity. Evaluation of a short, medium and long RT
compound in a labelꢀfree assay indicated a correlation between RT and functional effect. The
effects of long RT compound 9 on sleepꢀwake states indicated long RT was translated into
sustained inhibition of rapid eye movement (REM) in vivo. These results show that affinityꢀonly
driven selection would have resulted in mGlu PAMs with high values for k , but not necessarily
2
on
optimized RT, which is key to predict optimal efficacy in vivo.
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INTRODUCTION
Glutamate is an important neurotransmitter in the human central nervous system (CNS), where
it modulates synaptic responses by activating ionotropic glutamate receptors (iGlu) and
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metabotropic glutamate receptors (mGlu). The eight known mGlu receptors, which belong to
the class C G proteinꢀcoupled receptors (GPCRs), have been divided into three subclasses based
on sequence homology, signaling pathway and pharmacological profile: group I (mGlu1, 5),
2
group II (mGlu2, 3) and group III (mGlu4, 6, 7, 8). Structurally, class C GPCRs consist of a large
extracellular orthosteric binding domain, the soꢀcalled venus flytrap (VFT), a cysteineꢀrich
2,3
domain connecting the VFT to the sevenꢀtransmembrane (7TM) domain. Group II mGlu
receptors are coupled to the Gαi/o protein and are generally expressed presynaptically in the
4
periphery of the synapse where they inhibit the release of glutamate upon activation. The
activation of mGlu receptors has been shown to be a potential strategy for the treatment of
2
psychiatric diseases involving abnormal glutamatergic neurotransmission, such as schizophrenia
5
,6
and anxiety disorder. Drug discovery efforts include the development of positive allosteric
modulators (PAMs) for which subtypeꢀselectivity can be more easily obtained as they bind
7
,8
within the less conserved 7TM domain and have little or no intrinsic activity on their own. The
mGlu2 PAMs Nꢀ(4ꢀ(2ꢀMethoxyphenoxy)phenyl)ꢀNꢀ(2,2,2ꢀtrifluoroethylsulfonyl)pyridꢀ3ꢀ
9
10
ylmethylamine (1, LY487379) , Biphenylꢀindanone A (2, BINA) , Nꢀ(4ꢀ((2ꢀ(Trifluoromethyl)ꢀ
3ꢀHydroxyꢀ4ꢀ(Isobutyryl)phenoxy)methyl)benzyl)ꢀ1ꢀMethylꢀ1HꢀImidazoleꢀ4ꢀCarboxamide (3,
11
THIIC, also known as LY2607540) and 3ꢀcyanoꢀ1ꢀcyclopropylmethylꢀ4ꢀ(4ꢀphenylꢀpiperidinꢀ1ꢀ
12
yl)ꢀpyridineꢀ2(1H)ꢀone (4, JNJꢀ40068782) have been characterized both in vitro and in vivo. To
date, two mGlu PAMs have reached clinical trials. The development of 7ꢀmethylꢀ5ꢀ[3ꢀ
2
(
piperazinꢀ1ꢀylmethyl)ꢀ1,2,4ꢀoxadiazolꢀ5ꢀyl]ꢀ2ꢀ[[4ꢀ(trifluoromethoxy)phenyl]methyl]ꢀ3Hꢀ
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isoindolꢀ1ꢀone (5, AZD8529) by AstraZeneca was discontinued after a phase 2 schizophrenia
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trial in which it did not show clinical efficacy when tested as monotherapy.
Compound 5
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recently finished a phase II clinical trial to study smoking cessation in female smokers.
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Meanwhile, the Janssen/Addex mGlu PAM 1ꢀbutylꢀ3ꢀchloroꢀ4ꢀ(4ꢀphenylꢀ1ꢀpiperidinyl)ꢀ(1H)ꢀ
2
pyridone (6, JNJꢀ40411813/ADX71149) met the primary objectives of safety and tolerability in
an exploratory phase IIa study in schizophrenia. Moreover, patients treated with antipsychotics
who experience residual negative symptoms were identified as the subgroup that may potentially
benefit from addꢀon treatment with 6, although this is yet to be established in a formal proofꢀofꢀ
concept study. In addition, in a second phase IIa study with 6 as adjunctive therapy in patients
having major depressive disorder (MDD) with significant anxiety symptoms, 6 did not meet the
criterion for efficacy vs placebo. Although 6 showed efficacy signals on several anxiety and
depression measures, the primary end point was not met and overall the results did not suggest
efficacy for 6 as an adjunctive treatment for patients with MDD with significant anxious
1
6–22
symptoms.
To understand more about the key in vitro parameters that could drive in vivo efficacy, we
have focused on the ligand binding kinetics in addition to more classical in vitro parameters. It is
now becoming increasingly apparent that in vivo efficacy can depend on optimized kinetic
2
3
behavior. Indeed, in the last decade the concept of ligand binding kinetics has received an
2
4–27
increasing amount of interest.
Both the association and dissociation rate constants k and k
on
off
28
have been studied for various drug targets including GPCRs. The relevance of residence time
(
RT), defined as the reciprocal of the dissociation rate constant (RT = 1/k ), for in vivo efficacy
off
was retrospectively shown for multiple marketed GPCR drugs, such as the long acting M3
receptor antagonist tiotropium and fast dissociating D antagonists with lower sideꢀeffect
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burden.
Whereas dissociation rates have been the focus of most kinetic studies, association
rates have also been described to be important for fast drug action and high receptor
3
2–34
occupancy.
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Here, we report the synthesis and biological evaluation of a novel series of 7ꢀarylꢀ1,2,4ꢀ
triazolo[4,3ꢀa]pyridines, which evolved from a pyridone series (4 and 6) through a scaffold
3
5,36
hopping drugꢀdesign strategy previously described.
These compounds were characterized as
mGlu PAMs in a functional assay and additionally, radioligand binding experiments were
2
performed to determine their affinity and kinetic binding parameters. We demonstrate that the
differentiated binding kinetics result in a varied response in a labelꢀfree assay on whole cells. In
this way we have been able for the first time to obtain both structureꢀactivity/affinity
relationships and structureꢀkinetic relationships for a class C GPCR.
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CHEMISTRY
The target compounds 8−48 were prepared via Suzuki cross coupling of the corresponding 7ꢀ
halotriazolopyridines (49a,b; 50a-c and 51a,b) with an array of boronic esters (52a-v) as shown
in Scheme 1. The chemical structures of compounds 8−48 are shown in Tables 3ꢀ6).
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The key 7ꢀhalotriazolopyridines (49a,b; 50a-c and 51a,b) and nonꢀcommercially available
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7,38
boronic esters (52a-v) were prepared following the procedures previously described.
BIOLOGY
The potency of the compounds was determined by quantifying the increase in response of a
3
5
fixed EC glutamate concentration (4 µM) observed in a functional [ S]GTPγS binding assay.
20
The affinity of all compounds was determined in a full curve radioligand displacement assay in
the presence of 6 nM of the mGlu2 PAM 3ꢀ(Cyclopropylmethyl)ꢀ7ꢀ[(4ꢀphenylꢀ1ꢀ
piperidinyl)methyl]ꢀ8ꢀ(trifluoromethyl)ꢀ1,2,4ꢀtriazolo[4,3ꢀa]pyridine (7, JNJꢀ46281222), which
3
9
was radioactively labelled with tritium. In order to determine the kinetic parameters k and k ,
on
off
we performed a radioligand competition association assay characterized by the equations of
Motulsky and Mahan that describe the binding between two ligands competing for the same
40
receptor binding site. For this purpose, first the association rate constant k and dissociation
on
rate constant koff of 7 were determined in radioligand association and dissociation experiments
4
1
using a scintillation proximity assay (SPA). Kinetic parameters were determined at 28°C,
presumably these will increase at 37°C. All kinetic assays were performed in the presence of 1
mM glutamate; this condition induced a monophasic association process of the PAM, which is
3
9
required for the determination of the kinetic parameters of unlabeled PAMs. The presence of
39
glutamate was shown to not affect affinity and kinetic parameters of 7. Ultimately, three
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compounds with representative RT values were selected for comparison in a labelꢀfree,
impedance based assay (xCELLigence) which measures ligandꢀinduced responses on whole
cells. This assay provided further understanding of the influence of RT on the duration of PAMꢀ
induced receptor activation.
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RESULTS AND DISCUSSION
Kinetic rate constants kon and koff for 7 were similar when obtained in the association and
dissociation assays compared to the competition association assay (Table 1), which validated the
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use of the competition association assay for all other compounds. Kinetic K values were
D
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comparable to the values reported before.
Reference mGlu PAMs 2ꢀ5 (Table 2) showed moderate to good potency and affinity at the
2
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mGlu receptor, in agreement with previous reports.
Kinetic parameters k , k and RT
on off
2
were also determined. Reference compound 2 was shown to have a RT of 11.6 minutes. The
other reference PAMs had RTs in the same range, from 7.8 minutes for 5 to 17.3 minutes for 7
(Table 1).
The analysis of the triazolopyridines containing a substituted pyridine (Table 3) began with
1
elaboration of the R substitution. Compound 8 was taken as the starting point. Substitution of
1
chlorine at the R position by trifluoromethyl (9) resulted in an increase in potency, while affinity
remained the same. Introduction of methyl (10) or isoꢀpropyl (11) led to an approx. 10ꢀfold
decrease in affinity and potency. Interestingly both k and RT values were similar for 8, 10 and
on
1
1 but 2ꢀfold (k ) and approx. 3ꢀfold (RT) higher for 9. Substitution of fluorine by chlorine at
on
2
the R position resulted in increased potency and affinity for 12, 13 and 14. In both cases, the
1
PAMs with CF at R (9 and 13) showed modestly longer RTs of 30 and 23 minutes,
3
respectively, along with highest potency and affinity.
3
Exploration of the pyridine R revealed the importance of the position of the nitrogen.
Compound 15 showed decreased potency and affinity compared to 8. A similar trend was seen
for 16 that showed values comparable to those reported previously which revealed decreased
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potency and affinity compared to 9. Values for k followed this trend whereas RTs remained
on
the same for 15, but decreased from 30 to 11 minutes for 16. Subsequently the alkyl substituents
on the pyridine ring were evaluated. Substitution of methyl in 15 by ethyl in 17, led to an
increased potency and affinity. Addition of a methyl group on the 5ꢀposition of 16, did not
change affinity or potency in 18, nor were large changes seen in kinetic parameters. Introduction
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of the bulky and lipophilic isoꢀpropyl in 19 and 20 increased potency, affinity and k slightly.
on
Interestingly, the RT of 20 was reduced to only 5 minutes.
4
Substitution of the cyclopropylmethyl substituent at the R position by 2,2,2ꢀtrifluoroethyl (21)
resulted in a slight decrease in affinity, similar values for potency and RT, and a 5ꢀfold increase
4
in k compared to 8. Next, the ethoxymethyl substituent was evaluated at R (22), which yielded
on
a slight decrease in potency, a 10ꢀfold decrease in kon and similar values for affinity and RT
1
compared to 21. Substitution of F in 22 by CF at R did not yield a drastic increase in affinity
3
4
and potency (23). Together, 21ꢀ23 showed that a cyclopropyl methyl substituent at R is
4
preferred, as in all three cases the analogue bearing cyclopropyl methyl at R showed a higher
affinity (8 or 9).
The structure of substituted pyridine of 8 was changed to the short aliphatic isoꢀ or cyclopropyl
substituent, yielding 24ꢀ30 (Table 4). Potency and affinity of 24 were relatively low (pEC 6.65;
5
0
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pK 7.21). Changing the R substituent to CF (25) increased affinity and potency again (pEC
i
3
50
2
7
.03; pK 8.05). Substitution of the fluorine on R to chlorine (26) resulted in a
̴
3ꢀfold increase in
i
affinity and potency.
3
Substitution of iꢀPr at the R position by a cꢀPr induced an increase in affinity and potency in
2
all cases (27ꢀ29). As observed in Table 3, replacing F at R in 27 by Cl in 30 led to an increased
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affinity and potency and a concomitant increase in k . The data in Table 4 show that k values
on
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followed affinity, whereas RT values were all within a narrow range (8.2 – 11.9 min).
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In addition to the results obtained with the first subꢀseries of triazolopyridines 8ꢀ23, a 4ꢀ
methoxyꢀ3ꢀpyridine moiety connected by a methylene spacer was introduced, leading to
compounds 31-35 (Table 5). These molecules share a moderate to good potency and affinity
2
(
pEC50 6.8 – 7.8; pK 7.9 – 8.7). Removal of the fluorine at the R position (32) did not
i
significantly change affinity, potency or kinetic parameters when compared to 31. Changing the
ether linker in 31 and 32 to an amine in 33 and 34 resulted in an approx. 3ꢀfold increase in
affinity and potency. A moderate increase in k was observed and interestingly a 4ꢀ and 2ꢀfold
on
increase in RT for 33 and 34, respectively. This may be explained by the increased flexibility
given by the methylene bridge between the triazolopyridine core and the pyridine substituent. As
seen in many examples, substitution of CF in 34 to Cl in 35 led to a decrease in affinity, potency
3
and kinetic parameter values.
Reducing aromaticity and introducing more aliphatic and sp3 character can sometimes be
4
4
beneficial for physicochemical properties and drugꢀlikeness. In order to evaluate the effects of
aromaticity, the 4ꢀmethoxyꢀ3ꢀpyridyl substituent was replaced with a 4ꢀtetrahydropyranyl ring
(36, Table 6). This change resulted in a (pK ) and potency (pEC ) of 6.65 and 7.64, respectively,
i
50
1
which were increased in 37 by the introduction of CF in the R position. Compound 37 has a
3
7
ꢀ1
ꢀ1
high affinity (pK 8.28) and increased kon (2.0 x 10 M min ), however, contrary to the high
i
affinity, its RT (4.1 min) was the shortest found throughout this study. As seen in the first series
of ligands (Table 2), affinity and potency did not change by altering the linker atom from ether to
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amine (38, 39). Increasing the linker length by a single methylene unit resulted in an approx. 10ꢀ
fold loss in affinity, (40; pK 7.21). Values for k followed affinity, but RT values were all in the
i
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The tetrahydropyran moiety was then replaced by a cyclohexanol ring, leading to 41ꢀ48.
1
Again, compounds bearing CF at R (42, 46) showed better affinity and potency than their
3
chlorine analogues (41, 45). Molecule 42 not only showed a high potency (pEC 8.03), affinity
5
0
7
ꢀ1
ꢀ1
(
pK 8.66) and k (1.1 x 10 M min ), but also a longer RT of 31 minutes. Its cisꢀdiastereomer
i on
43 showed a 3ꢀfold reduction in potency (pEC 7.38) and affinity (pK 8.03), a 2ꢀfold reduction
5
0
i
6
ꢀ1
ꢀ1
in kon (5.7 x 10 M min ) and a small reduction in RT (17 min). Introduction of a cꢀPr
3
substituent at R (44) led to an increase in potency (pEC 8.95), affinity (pK 9.05) and k (1.4 x
50
i
on
7
ꢀ1
ꢀ1
10 M min ), and also to a prolonged RT of 50 minutes, which was the highest value observed
3
in this study. It may be that the cyclopropyl at R directs the hydrophilic hydroxyl into a better
4
position for interaction with the receptor. Changing R from cꢀPr to CF (45) did not change any
3
parameter compared to 41. Changing the linker group from amine in 43 to ether in 46 did not
affect affinity and potency. However, RT seemed to be somewhat increased. Differences
between diastereomers were seen again in 47 and 48. Whereas the potencies were similar,
affinity was decreased 3ꢀfold and k remained similar, while RT was prolonged by 2ꢀfold when
on
moving from a trans to cis configuration.
1
In general, CF was shown to be the preferred substituent at R . Compounds bearing this
3
substituent typically had a higher potency and affinity. Interestingly, all PAMs with a RT longer
than 20 minutes bear this moiety, including 44 with the longest RT seen in this study (50 min).
On the other hand, molecule 37 had the shortest RT (4.1 min) and also contains the CF group,
3
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indicating that CF in itself is not the driver for long RT. At the R position chlorine was shown
3
to be the best substituent for high potency and affinity. PAMs bearing a substituted pyridine
revealed the importance of its substitutions in combination with the position of the nitrogen in
the ring for potency, affinity and kinetic parameters.
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The rather small differences in RT induced by substitutions at the R and R position at the
central triazolopyridine core indicated that this region does not induce large differences in RT.
On the other hand, small differences in the distal tail of the compounds induced large differences
4
in RT. As we have shown previously, this triazolopyridine series binds with the scaffold and R
substituent in the deepest part of the receptor, whilst the distal tail points to the extracellular
3
9
side. These differences in RT may be explained by the tight receptorꢀligand interactions of the
triazolopyridine core compared to looser interactions on the extracellular side. Certain molecules
may trap the more flexible ECL2 (or the tops of TMs 3 and 5) in conformations leading to longer
RT. Such induced fit modulation of RT can involve ordering of loops to partially block the
binding pocket and prevent ligand escape. This mechanism has been seen for multiple targets
from different classes, amongst which the 5ꢀHT2B receptor as reported in the recently published
2
4,45
LSDꢀbound crystal structure.
Selectivity of this series of mGlu PAMs for other members of
2
the mGlu family was good. Representatives (12, 27, 37 and 45) showed no activity at mGlu1, 3,
4, 5, 6, 7 or 8, see Supporting Information table S1.
In order to compare the kinetic parameters and affinity, a kinetic map was generated (Fig. 2).
In this map k (xꢀaxis), k (yꢀaxis) and K (diagonal lines) were plotted. Whereas k values
on
off
D
off
ꢀ1
were all within a small range of about one order of magnitude between 0.020 min (44) and
ꢀ1
0
.241 min (37), k values were more diverse and were spread over almost two orders of
on
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ꢀ1
ꢀ1
7
ꢀ1
ꢀ1
magnitude between 1.7 x 10 M min (40) to 7.7 x 10 M min (28 and 29). The molecules
were plotted per subgroup, as presented in the different tables. This revealed that 8ꢀ23 and 24ꢀ30
7
ꢀ1
ꢀ1
7
ꢀ1
ꢀ
were mostly diverse in onꢀrates (8ꢀ23: 0.17 – 6.2 x 10 M min ; 24ꢀ30: 0.48 – 7.7 x 10 M min
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1
ꢀ1
)
, whereas 31ꢀ35 and 36ꢀ48 were more diverse in offꢀrates (31ꢀ35: 0.044 – 0.17 min ; 36ꢀ48:
ꢀ1
0.020 – 0.24 min ), indicating that an increased affinity between close analogues may be
achieved by optimizing one kinetic parameter at a time. Interestingly, the five reference mGlu2
PAMs 2ꢀ5 and 7, which are very diverse in structure and affinity, differed mostly in terms of onꢀ
6
ꢀ1
ꢀ1
ꢀ1
rate (0.21 – 4.6 x 10 M min ), whereas offꢀrates were very close (0.086 – 0.13 min ). As
expected, the compounds with the highest affinity either have a long RT (44) or a fast k (28ꢀ
on
2
9). Ideally, these optimized parameters may be combined leading to an mGlu PAM with even
2
more increased affinity, which would be represented in the top right corner of the kinetic map.
For further comparison, the compounds were arbitrarily divided in short RT (RT < 10 min),
medium RT (10 – 20 min) and long RT (> 20 min) (Fig. 2).
To gain more insight in the relationships between the different parameters studied, correlation
plots were made (Fig. 3). First, the affinity (pK ) and potency (pEC ) were shown to be linearly
i
50
correlated (Fig. 3A). Secondly, the affinity obtained from the classical radioligand displacement
assay (pK ) and affinity obtained from kinetic parameters (pK ), were shown to be correlated
i
D
(Fig. 3B). This further supported the use of the kinetic SPA radioligand binding assay. Long RT
PAMs were shown to share a high affinity and potency, but by itself a high potency or affinity
does not predict a long RT (Fig. 3A, 3B, S1). Then, the dissociation rate constants (pk ) were
off
plotted against the kinetic affinity data (pK ) (Fig. 3C), which did not show correlation.
D
Interestingly, the association rate constants (log k ) were strongly correlated to affinity (pK )
on
D
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(
Fig 3D). These parameters were both within a similar range of roughly two orders of magnitude
for the compounds of our series. Interestingly, although having different structures and in some
cases lower affinities and kon values, the reference mGlu PAMs (2ꢀ5) also fitted well in this
2
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correlation (Fig. 3D). This indicated that this correlation has likely revealed a general trend for
PAMs and how they bind at the mGlu receptor.
2
Correlations between kinetic parameters and affinity have been made before. Most of these
studies found a correlation between k values and affinity or efficacy, for instance on the M and
off
3
4
6–48
A2A receptors.
However, a correlation between association rate constants and affinity, as was
shown here for mGlu PAMs, has also been observed before. Exemplary GPCRs are the OX2
2
4
9,50
51,52
receptor and β ꢀadrenoreceptors,
and other targets such as the hERG channel.
These
2
correlations are receptorꢀspecific and may therefore be caused by differences in receptor
structure, dynamics, or local environment of the receptor.
Recent simulation studies have stipulated the importance of fast association rates for receptor
3
3,53
occupancy and drug dosing.
One of the mechanisms which may be responsible for these
effects is receptor rebinding, which is described as the binding of freshly dissociated ligands
5
4
from the local environment of the receptor. These high local concentrations may lead to a
prolonged activity of the drug, even when concentrations within the effect compartment have
5
5
fallen below therapeutic levels. For the mGlu receptor, rebinding may play a role. The
2
physiological synaptic environment of the receptor may permit more rebinding as its interstitial
56,57
location will result in less diffusion and therefore higher local ligand concentrations.
Another
reason for receptor rebinding would be binding of compound to or in the cell membrane close to
the receptor which may then act as a repository and facilitate the approach of the compound to
56
the receptor. This would, however, require a correlation between lipophilicity and association
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rate constants, as lipophilic compounds are more likely to bind to the cell membrane. Since the
plot in figure 3E shows no such correlation for the studied mGlu PAMs, membrane binding is
2
not likely to be involved for these compounds.
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xCELLigence
We used a labelꢀfree, impedanceꢀbased technology (xCELLigence) to evaluate whether a long
receptor binding RT leads to a prolonged functional activity. This method is based on the
5
8
measurement of cellular impedance, expressed as Cell Index, and enables realꢀtime recordings.
Responses induced by compound addition can thus be measured and quantified. Earlier studies
5
9
have shown that results obtained were comparable to more classical endpoint assays. As the
assay is performed on whole cells at 37°C it is considered a valuable translational step towards in
60
vivo experiments. Endogenous glutamate levels at time of experiments were around 90 µM.
Compoundꢀinduced responses therefore represent the PAM effects. As a control, reference
compound 7 was used to check the response on CHOꢀK1 WT cells compared to CHOꢀ
K1_hmGlu cells (Fig. S2). No response was found on the WT cells, indicating that the
2
responses were mGlu receptorꢀmediated. For further experiments we selected compounds with
2
diversity in RT (measured at 28°C): 37 (4.1 min), its close analogue 39 with 3ꢀ4 ꢀfold longer RT
(
15 min) and 44 with again a 3ꢀfold longer RT compared to 39 (50 min; Fig. 4AꢀD). We
determined their potency in the labelꢀfree assay using CHOꢀK1_hmGlu cells and found pEC50
2
values of 7.05 ± 0.04, 7.73 ± 0.23 and 7.95 ± 0.12, respectively. Then we performed a washꢀout
assay, in order to evaluate differences in compoundꢀinduced responses after washing. This
enabled us to trace the remaining response exerted by only receptorꢀbound compound. To this
end we first determined the EC concentrations of short RT 37, medium RT 39 and long RT 44,
8
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which were 600, 200 and 60 nM, respectively. Then, cells were stimulated with this EC80
concentration and just before maximal response was reached, after 5 minutes, cells were washed
and fresh medium was added (Fig. 4EꢀG). By comparing these traces with control traces of
unwashed wells, we evaluated the differences in responses between 37, 39 and 44 as the ratio
between the AUC of washed and unwashed wells (Fig. 4H). The functional effect of 37 and 39
were partially lost in this experimental setꢀup, while the effect of 44 remained almost unchanged.
The washꢀout assay showed a significant difference between short RT 37 and long RT 44. The
value for 39 was in between those of 37 and 44, in agreement with its RT. Together, these data
showed that a longer RT also leads to an increased functional effect under nonꢀequilibrium
conditions and that these parameters are positively correlated. Since equilibrium conditions are
not always present in physiological conditions, these findings may be considered a translational
step from in vitro towards in vivo experiments.
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In vivo sleep-wake effects
Glutamate neurotransmission plays a key role in sleepꢀwake mechanisms and these processes
have translational value for central activity and target engagement. Previous in vivo studies have
repeatedly shown that positive allosteric modulation of the mGlu receptor results in distinct
2
changes in rodent sleepꢀwake organization, more specifically in a doseꢀdependent reduction in
6
1,62
soꢀcalled Rapid Eye Movement Sleep or REM sleep.
Changes in sleepꢀwake states in the rat
are of a dynamic nature, i.e. with more frequent transitions between sleepꢀwake states as
compared to humans: therefore the effects of mGlu PAM compounds on REM sleep are
2
expressed as averages over for example 2 hour or 4 hours periods after compound
administration. In order to investigate whether the different RT values are reflected in the sleepꢀ
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wake related pharmacodynamic readꢀout, sleepꢀwake analysis of 9 and 20 (selected as examples
with long versus short in vitro RT, respectively, yet having appropriate and similar
pharmacokinetic behavior) was even done on a 5 minute basis to optimally capture sleep changes
in function of time. All methods, (circadian) timings and analyses otherwise were similar to
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6
2
those reported earlier by Ahnaou et al. (2016).
Figure 5 shows the effects of 9 and 20 on REM sleep reduction in rats for 24 consecutive 5ꢀ
min periods after a single oral administration of 1, 3, and10 mg/kg and 20% CD vehicle control.
The first 6 consecutive 5ꢀminute periods can be discarded due to the confounding arousing effect
of the oral administration procedure per se, the animal returning to undisturbed REM sleep
baseline values after about 30 minutes. Molecule 9 showed a longer in vitro RT of 30 minutes
whilst for 20 it was 5 minutes. Correspondingly, at the higher dose of 10 mg/kg, the effect of 9
on REM reduction seemed more immediate and constant compared to 20. For both compounds
no effects on total sleep were seen (Fig. S3).
Compound 9 and 20 showed similar in vitro binding affinities with pK values of 8.59 and
i
8.44, respectively. At 10 mg/kg, estimated free levels in brain 1 hour postꢀadministration are 5
and 3.5 ng/g for 9 and 20, respectively. Hence the two molecules show similar affinity and
roughly similar range of free concentrations in brain. While the underlying mechanism causing
this different in vivo effect is still to be elucidated, this finding provides a first hint that different
RT’s may impact the in vivo effect. The interplay of RT with elimination/clearance from brain is
important for the resulting in vivo effect, but with similar levels of 9 and 20 after 1 hour, it seems
that RT may play a role in this case. To strengthen these observations, future experiments could
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include the synthesis of PET ligands with different residence times and further in vivo studies
with a different endpoint such as cognition.
While potent GPCR antagonists can act via a long RT with expected improved clinical efficacy
there is limited published data assessing the relationship between agonist/PAM RT and in vivo
efficacy. Agonist responses are usually regulated by receptor desensitization and internalization,
which can act to limit the effect and duration of receptor signaling. PAMs do not activate
receptors in the absence of agonist, and hence allosteric modulators with prolonged action may
not be negated by internalization and loss of function due to longer RT of the PAMꢀreceptor
complex.
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CONCLUSIONS
In this study we described a series of 7ꢀarylꢀ1,2,4ꢀtriazolo[4,3ꢀa]pyridines with potent mGlu2
PAM activity and a high affinity. An SPA radioligand binding assay was developed to study the
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kinetic parameters of these compounds. We showed that association rate constants range within
three orders of magnitude, whereas RT values are more confined, from 4.1 minutes (37) up to 50
minutes (44). Correlation plots between affinity and kinetic parameters revealed a strong
correlation between affinity and association rate constants, whereas no such correlation was
found between RT and affinity.
Structureꢀkinetics relationships were explored in addition to the more traditional structureꢀ
1
activity/affinity relationships analysis. We learned that a CF substituent at R increased potency
3
and affinity and was also important for RT, but did not lead to a long RT per se as shown by its
2
presence in 37 (‘short’ RT) and 44 (‘long’ RT). Chlorine was the preferred substituent for R , it
increased potency and affinity but not RT. Differences in the distal tail of the molecules, such as
small changes in the pyridine moiety or single substitutions to the cyclohexanol, induced the
biggest differences in RT. This was exemplified by the introduction of cꢀPr in 44, leading to a 3ꢀ
fold increase in RT compared to 43.
To evaluate whether a long RT also leads to a prolonged functional effect, 37, 39 and 44 were
evaluated in a washꢀout assay using the labelꢀfree impedance based xCELLigence technique.
The response of 44 on whole cells at physiological temperature remained more sustained after
washing than that of 37 and 39. These data suggested that an increased lifetime of the receptorꢀ
ligand complex (RT) is correlated to an increased functional effect under nonꢀequilibrium
conditions.
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Together, these results show for the first time for a class C GPCR that optimization of ligand
binding kinetics in addition to potency and affinity is possible. Given that pure PAMs do not
exert an effect in the absence of glutamate and are thus less likely to induce onꢀtarget toxicity, a
long RT seems a promising strategy for these ligands. As rebinding may be involved in mGlu2
receptor occupancy, a RT for 44 in our system of 50 minutes may be prolonged in the interstitial
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0
synaptic environment. Without any onꢀtarget toxicity occurring, the design of mGlu PAMs with
2
even longer RTs may be a logical next step. Lastly, a first attempt was made to relate the
compound’s RT to its in vivo pharmacodynamic effect. Ultimately, this study may contribute to
the development of compounds with a high affinity and efficacy in vitro and in vivo, not only for
the mGlu receptor but also for other GPCRs.
2
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EXPERIMENTAL SECTION
Chemistry
Unless otherwise noted, all reagents and solvents were obtained from commercial suppliers
and used without further purification. Thin layer chromatography (TLC) was carried out on silica
gel 60 F254 plates (Merck). Flash column chromatography was performed on silica gel, particle
size 60 Å, mesh of 230−400 (Merck) under standard techniques. Microwave assisted reactions
were performed in a singleꢀmode reactor, Biotage Initiator Sixty microwave reactor (Biotage), or
in a multimode reactor, MicroSYNTH Labstation (Milestone, Inc.). Nuclear magnetic resonance
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(
NMR) spectra were recorded with either a Bruker DPXꢀ400 or a Bruker AVꢀ500 spectrometer
Bruker AG) with standard pulse sequences, operating at 400 and 500 MHz, respectively, using
(
CDCl and DMSOꢀd as solvents. Chemical shifts (δ) are reported in parts per million (ppm)
3
6
downfield from tetramethylsilane (δ = 0). Coupling constants are reported in hertz. Splitting
patterns are defined by s (singlet), d (doublet), dd (double doublet), t (triplet), q (quartet), quin
(
quintet), sex (sextet), sep (septet), or m (multiplet). Liquid chromatography combined with mass
spectrometry (LCMS) was performed on either a HP 1100 HPLC system (Agilent Technologies)
or Advanced Chromatography Technologies system composed of a quaternary or binary pump
with degasser, an autosampler, a column oven, a diode array detector (DAD), and a column as
specified in the respective methods below. Flow from the column was split to a MS
spectrometer. The MS detector was configured with either an electrospray ionization source or
an ESCI dual ionization source (electrospray combined with atmospheric pressure chemical
ionization). Nitrogen was used as the nebulizer gas. Data acquisition was performed with
MassLynxꢀOpenlynx software or with ChemsationꢀAgilent data browser software. Melting point
values are peak values and were obtained with experimental uncertainties that are commonly
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associated with this analytical method. Melting points were determined in open capillary tubes
on a Mettler FP62 apparatus with a temperature gradient of 10 °C/min. Maximum temperature
was 300 °C.
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Purities of all new compounds were determined by analytical RP HPLC using the area
percentage method on the UV trace recorded at a wavelength of 254 nm, and compounds were
found to have ≥95% purity unless otherwise specified.
8-Chloro-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (8). To a stirred suspension of 50a (1.7 g, 5.09
mmol) and 52a (2.1 g, 6.12 mmol) in a saturated aqueous solution of NaHCO (18 mL) and 1,4ꢀ
3
dioxane (36 mL) was added Pd(PPh ) (0.589 g, 0.51 mmol). The mixture was heated at 150 °C
3 4
for 10 min under microwave irradiation. The mixture was cooled to room temperature and
filtered through a Celite pad. The filtrate was diluted with water (20 mL) and extracted with
EtOAc (2 × 15 mL). The organic layer was washed with brine (15 mL), dried over anhydrous
Na SO , and concentrated in vacuo. The crude was purified by flash column chromatography
2
4
(silica gel, EtOAc in DCM, 0/100 to 20/80) to give the desired product 8 as a white solid (1.3 g,
1
60%). Mp 207.2 °C. H NMR (500 MHz, CDCl ) δ ppm 0.32ꢀ0.42 (m, 2H), 0.61ꢀ0.69 (m, 2H),
3
1.17ꢀ1.28 (m, 1H), 2.54 (s, 3H), 2.55 (s, 3H), 3.13 (d, J = 6.9 Hz, 2H), 6.87 (d, J = 6.9 Hz, 1H),
6.92 (t, J = 8.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 7.16 (d, J = 8.4 Hz, 1H), 7.25 (d, J = 9.2 Hz,
+
1H), 7.41 (dd, J = 11.3, 1.7 Hz, 1H), 7.98 (d, J = 6.9 Hz, 1H). LC−MS m/z 423 [M + H] , t =
R
2
.86 min.
-Trifluoromethyl-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (9). Starting from 49a (0.150 g, 0.54 mmol) and
8
52a (0.242 g, 0.707 mmol) and following the procedure described for 8, compound 9 was
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obtained as a white solid (0.139 g, 56%). H NMR (400 MHz, CDCl ) δ ppm 0.31ꢀ0.43 (m, 2H),
3
0
.61ꢀ0.70 (m, 2H), 1.16ꢀ1.30 (m, 1H), 2.53 (s, 3H), 2.55 (s, 3H), 3.15 (d, J = 6.7 Hz, 2H), 6.79
(
d, J = 7.2 Hz, 1H), 6.89 (t, J = 8.3 Hz, 1H), 7.01 (d, J = 8.3 Hz, 1H), 7.05 (br d, J = 8.6 Hz, 1H),
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
7
.14 (d, J = 8.3 Hz, 1H), 7.22 (dd, J = 10.9, 2.1 Hz, 1H), 8.11 (d, J = 7.2 Hz, 1H). LC−MS m/z
+
457 [M + H] , t = 3.03 min.
R
8-Methyl-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (10). Starting from 51a (0.250 g, 1.127 mmol)
and 52a (0.464 g, 1.35 mmol) and following the procedure described for 8, compound 10 was
1
obtained as a white solid (0.103 g, 23%). Mp 147.4 °C. H NMR (500 MHz, CDCl ) δ ppm 0.32
3
ꢀ
0.39 (m, 2H), 0.60 ꢀ 0.67 (m, 2H), 1.16 ꢀ 1.29 (m, 1H), 2.55 (s, 6H), 2.65 (s, 3H) 3.11 (d, J =
6.65 Hz, 2H), 6.78 (d, J = 7.22 Hz, 1H), 6.93 (t, J = 8.38 Hz, 1H), 7.00 (d, J = 8.38 Hz, 1H), 7.07
(d, J = 8.38 Hz, 1H), 7.13 (d, J = 8.09 Hz, 1H), 7.23 (dd, J = 11.27, 2.02 Hz, 1H), 7.89 (d, J =
+
6.94 Hz, 1H). LC−MS m/z 403 [M + H] , t = 2.07 min.
R
8-Cyclopropyl-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (11). Starting from 51b (0.230 g, 0.93 mmol) and
52a (0.382 g, 1.11 mmol) and following the procedure described for 8, compound 11 was
1
obtained as a white solid (0.269 g, 68%). Mp 166.4 °C. H NMR (400 MHz, CDCl ) δ ppm 0.29
3
ꢀ
0.38 (m, 2H), 0.58 ꢀ 0.67 (m, 2H), 0.90 ꢀ 1.00 (m, 2H), 1.14 ꢀ 1.31 (m, 1H), 1.55 ꢀ 1.70 (m, 2H)
2.10 (tt, J=8.64, 5.46 Hz, 1H), 2.54 (s, 3H), 2.55 (s, 3H), 3.06 (d, J=6.70 Hz, 2H), 6.73 (d,
J=7.17 Hz, 1H), 6.93 (t, J=8.44 Hz, 1H), 7.00 (d, J=8.32 Hz, 1H), 7.13 (d, J=8.32 Hz, 1H), 7.15
ꢀ
7.20 (m, 1H), 7.32 (dd, J = 11.21, 1.97 Hz, 1H), 7.80 (d, J = 7.17 Hz, 1H). LC−MS m/z 429 [M
+
+
H] , t = 2.24 min.
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-Chloro-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
chlorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (12). Starting from 50a (0.250 g, 0.75 mmol) and
5
2b (0.385 g, 0.75 mmol) and following the procedure described for 8, compound 12 was
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8
9
0
1
2
3
4
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6
7
8
9
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obtained as a white solid (0.100 g, 70%). Mp 182.7 °C. H NMR (400 MHz, CDCl ) δ ppm 0.31
3
ꢀ 0.41 (m, 2H), 0.57 ꢀ 0.70 (m, 2H), 1.21 ꢀ 1.33 (m, 1H), 2.51 (s, 3H), 2.56 (s, 3H), 3.13 (d, J =
6
.94 Hz, 2H), 6.82 (d, J = 8.55 Hz, 1H), 6.87 (d, J = 6.94 Hz, 1H), 7.03 (d, J = 8.32 Hz, 1H),
7
.16 (d, J = 8.09 Hz, 1H), 7.37 (dd, J = 8.55, 2.31 Hz, 1H), 7.66 (d, J = 2.31 Hz, 1H), 7.97 (d, J
+
= 7.17 Hz, 1H). LC−MS m/z 439 [M + H] , t = 3.08 min.
R
8
-Trifluoromethyl-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
chlorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (13). Starting from 49a (0.2 g, 0.73 mmol) and
52b (0.26 g, 0.72 mmol) and following the procedure described for 8, compound 13 was
1
obtained as a white solid (0.217 g, 61%). Mp > 300ºC. H NMR (400 MHz, CDCl ) δ ppm 0.34 ꢀ
3
0
.41 (m, 2H), 0.62 ꢀ 0.70 (m, 2H), 1.16 ꢀ 1.30 (m, 1H), 2.50 (s, 3H), 2.56 (s, 3H), 3.15 (d, J =
6
.70 Hz, 2H), 6.79 (d, J = 8.09 Hz, 2H), 7.03 (d, J = 8.09 Hz, 1H), 7.11 ꢀ 7.19 (m, 2H), 7.49 (d, J
+
=
2.31 Hz, 1H), 8.10 (d, J = 7.17 Hz, 1H). LC−MS m/z 473 [M + H] , t = 3.22 min.
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8-Methyl-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
chlorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (14). Starting from 60a (0.250 g, 0.127 mmol)
and 52b (0.464 g, 1.35 mmol) and following the procedure described for 8, compound 14 was
1
obtained as a white solid (0.107 g, 22%). Mp 121 °C. H NMR (500 MHz, CDCl ) δ ppm 0.36
3
(
q, J = 5.01 Hz, 2H), 0.58 ꢀ 0.67 (m, 2H), 1.17 ꢀ 1.30 (m, 1H), 2.52 (s, 3H), 2.56 (s, 3H), 2.64 (s,
3
8
1
H), 3.11 (d, J = 6.65 Hz, 2H), 6.78 (d, J = 6.94 Hz, 1H), 6.83 (d, J = 8.67 Hz, 1H), 7.02 (d, J =
.38 Hz, 1H), 7.13 (d, J = 8.09 Hz, 1H), 7.18 (dd, J = 8.38, 2.02 Hz, 1H), 7.49 (d, J = 2.31 Hz,
+
H), 7.89 (d, J = 6.94 Hz, 1H). LC−MS m/z 419 [M + H] , t = 2.23 min.
R
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-Chloro-3-cyclopropylmethyl-7-{4-[(2-methylpyridin-4-yl)oxy]-3-
fluororophenyl}[1,2,4]triazolo[4,3-a]-pyridine (15). Starting from 50a (1.71 g, 5.13 mmol) and
5
2c (1.4 g, 5.67 mmol) and following the procedure described for 8, compound 15 was obtained
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
as a white solid (0.100 g, 70%). Mp > 300ºC. H NMR (500 MHz, CDCl ) δ ppm 0.34 ꢀ 0.41 (m,
3
2H), 0.63 ꢀ 0.70 (m, 2H), 1.18 ꢀ 1.28 (m, 1H), 2.55 (s, 3H), 3.14 (d, J = 6.65 Hz, 2H), 6.73 (dd, J
=
5.78, 2.31 Hz, 1H), 6.78 (d, J = 2.31 Hz, 1H), 6.91 (d, J = 7.22 Hz, 1H), 7.27 ꢀ 7.34 (m, 1H),
7
.36 ꢀ 7.41 (m, 1H), 7.45 (dd, J = 10.69, 2.02 Hz, 1H), 8.01 (d, J = 6.94 Hz, 1H), 8.41 (d, J =
+
5.78 Hz, 1H). LC−MS m/z 409 [M + H] , t = 2.57 min.
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8
-Trifluoromethyl-3-cyclopropylmethyl-7-{4-[(2-methylpyridin-4-yl)oxy]-3-
fluororophenyl}[1,2,4]triazolo[4,3-a]-pyridine (16). Starting from 49a (1.4 g, 5.08 mmol) and
52c (1.84 g, 5.59 mmol) and following the procedure described for 8, compound 16 was obtained
1
as a white solid (0.233 g, 10%). Mp 194.6ºC. H NMR (500 MHz, CDCl ) δ ppm 0.34 ꢀ 0.44 (m,
3
2
H), 0.61 ꢀ 0.73 (m, 2H), 1.18 ꢀ 1.29 (m, 1H), 2.55 (s, 3H), 3.17 (d, J = 6.66 Hz, 2H), 6.70 (dd, J
5.8, 2.6 Hz, 1H), 6.76 (d, J = 2.3 Hz, 1H), 6.83 (d, J = 7.2 Hz, 1H), 8.20 (br d, J = 8.4 Hz, 1H),
=
7
.23 ꢀ 7.31 (m, 2H), 8.14 (d, J = 7.2 Hz, 1H), 8.41 (d, J = 5.8 Hz, 1H). LC−MS m/z 443 [M +
+
H] , t = 2.70 min.
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8
-Chloro-3-cyclopropylmethyl-7-{4-[(2-ethylpyridin-4-yl)oxy]-3-
fluororophenyl}[1,2,4]triazolo[4,3-a]-pyridine (17). Starting from 50a (0.26 g, 0.78 mmol) and
52d (0.293 g, 0.857 mmol) and following the procedure described for 8, compound 17 was
1
obtained as a white solid (0.316 g, 96%). Mp > 300ºC. H NMR (500 MHz, CDCl ) δ ppm 0.31 ꢀ
3
0
.43 (m, 2H), 0.60 ꢀ 0.72 (m, 2H), 1.15 ꢀ 1.29 (m, 1H), 1.31 (t, J = 7.7 Hz, 3H), 2.82 (q, J = 7.6
Hz, 2H), 3.14 (d, J = 6.6 Hz, 2H), 6.72 (dd, J = 5.8, 2.3 Hz, 1H), 6.81 (d, J = 2.3 Hz, 1H), 6.91
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(
d, J = 6.9 Hz, 1H), 7.31 (t, J = 8.2 Hz, 1H), 7.35 ꢀ 7.42 (m, 1H), 7.45 (dd, J = 10.7, 2.0 Hz, 1H),
+
8
.01 (d, J = 6.9 Hz, 1H), 8.44 (d, J = 5.8 Hz, 1H). LC−MS m/z 423 [M + H] , t = 2.04 min.
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8-Trifluoromethyl-3-cyclopropylmethyl-7-{4-[(2,6-dimethylpyridin-4-yl)oxy]-3-
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fluororophenyl}[1,2,4]triazolo[4,3-a]-pyridine (18). Starting from 49a (0.185 g, 0.67 mmol)
and 52e (0.3 g, 0.874 mmol) and following the procedure described for 8, compound 18 was
1
obtained as a white solid (0.100 g, 33%). Mp 232.5ºC. H NMR (500 MHz, CDCl ) δ ppm 0.24 ꢀ
3
0
.50 (m, 2H) 0.55 ꢀ 0.79 (m, 2H) 1.05 ꢀ 1.39 (m, 1H) 2.50 (s, 6H) 3.17 (d, J=6.70 Hz, 2H) 6.56
(s, 2H) 6.84 (d, J=7.17 Hz, 1H) 7.16 ꢀ 7.21 (m, 1H) 7.22 ꢀ 7.29 (m, 2H) 8.14 (d, J=7.17 Hz, 1 H).
+
LC−MS m/z 457 [M + H] , t = 2.94 min.
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8-Chloro-3-cyclopropylmethyl-7-{4-[(2-cyclopropylpyridin-4-yl)oxy]-3-
fluororophenyl}[1,2,4]triazolo[4,3-a]-pyridine (19). Starting from 50a (0.103 g, 0.310 mmol)
and 52f (0.22 g, 0.310 mmol) and following the procedure described for 8, compound 19 was
1
obtained as a white solid (0.06 g, 45%). Mp > 300ºC. H NMR (500 MHz, CDCl ) δ ppm 0.38
3
(
q, J = 5.11 Hz, 2H), 0.62 ꢀ 0.71 (m, 2H), 0.95 ꢀ 1.09 (m, 4H), 1.16 ꢀ 1.29 (m, 1H), 1.94 ꢀ 2.03
m, 1H), 3.14 (d, J = 6.65 Hz, 2H), 6.66 (dd, J = 5.64, 2.46 Hz, 1H), 6.78 (d, J = 2.31 Hz, 1H),
(
6.90 (d, J = 6.94 Hz, 1H), 7.28 ꢀ 7.33 (m, 1H), 7.34 ꢀ 7.40 (m, 1H), 7.44 (dd, J = 10.69, 2.02 Hz,
+
1
H), 8.00 (d, J = 6.94 Hz, 1H), 8.35 (d, J = 5.78 Hz, 1H). LC−MS m/z 435 [M + H] , t = 4.51
R
min.
8-Trifluoromethyl-3-cyclopropylmethyl-7-{4-[(2-cyclopropylpyridin-4-yl)oxy]-3-
fluororophenyl}[1,2,4]triazolo[4,3-a]-pyridine (20). Starting from 49a (0.2 g, 0.725 mmol) and
5
2f (0.283 g, 0.798 mmol) and following the procedure described for 8, compound 20 was
1
obtained as a white solid (0.058 g, 17%). Mp 211.1ºC. H NMR (500 MHz, CDCl ) δ ppm 0.32ꢀ
3
0
.45 (m, 2H), 0.53 ꢀ 0.75 (m, 2H), 0.96 ꢀ 1.03 (m, 2H), 1.02 ꢀ 1.08 (m, 2H), 1.16 ꢀ 1.30 (m, 1H),
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.91 ꢀ 2.03 (m, 1H), 3.16 (d, J = 6.7 Hz, 2H), 6.63 (dd, J = 5.8, 2.3 Hz, 1H), 6.75 (d, J = 2.3 Hz,
H), 6.83 (d, J = 7.2 Hz, 1H), 7.15ꢀ7.22 (m, 1H), 7.22 ꢀ 7.31 (m, 2H), 8.15 (d, J = 6.9 Hz, 1H),
+
.35 (d, J = 5.5 Hz, 1H). LC−MS m/z 469 [M + H] , t = 3.31 min.
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11
12
13
14
15
16
17
18
19
20
21
22
23
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41
42
43
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45
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47
48
49
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51
52
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57
58
59
60
8
-Chloro-3-(2,2,2-trifluoroethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (21). Starting from 56c (0.2 g, 0.55 mmol) and
5
2a (0.228 g, 0.664 mmol) and following the procedure described for 8, compound 21 was
1
obtained as a white solid (0.032 g, 13%). Mp 164.5ºC. H NMR (500 MHz, CDCl ) δ ppm 2.53
3
(s, 3H), 2.55 (s, 3H), 4.11 (q, J = 9.9 Hz, 2H), 6.93 (t, J = 8.3 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H),
7
.02 (d, J = 8.3 Hz, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.23 ꢀ 7.28 (m, 1H), 7.42 (dd, J = 11.1, 2.1 Hz,
+
1
H), 8.01 (d, J = 7.2 Hz, 1H). LC−MS m/z 451 [M + H] , t = 3.24 min.
R
8
-Chloro-3-(ethoxymethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (22). Starting from 50b (0.4 g, 1.19 mmol) and
5
2a (0.488 g, 1.42 mmol) and following the procedure described for 8, compound 22 was
1
obtained as a white solid (0.310 g, 61%). Mp 180.3ºC. H NMR (500 MHz, CDCl ) δ ppm 1.23
3
(
t, J = 6.94 Hz, 3H), 2.54 (s, 3 H), 2.55 (s, 3H), 3.58 (q, J = 6.94 Hz, 2H), 5.10 (s, 2H), 6.87 ꢀ
6.95 (m, 2H), 7.02 (d, J = 8.09 Hz, 1H), 7.16 (d, J = 8.09 Hz, 1H), 7.22 ꢀ 7.26 (m, 1H), 7.42 (dd,
+
J = 11.27, 2.31 Hz, 1H), 8.25 (d, J=7.22 Hz, 1H). LC−MS m/z 427 [M + H] , t = 2.77 min.
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8
-Trifluoromethyl-3-(ethoxymethyl-7-{4-[(2,6-dimethylpyridin-3-yl)oxy]-3-
fluorophenyl}[1,2,4]triazolo[4,3-a]-pyridine (23). Starting from 49b (0.150 g, 0.536 mmol)
and 52a (0.239 g, 0.697 mmol) and following the procedure described for 8, compound 23 was
1
obtained as a white solid (0.150 g, 61%). Mp 142.2ºC. H NMR (500 MHz, CDCl ) δ ppm 1.23
3
(
t, J=7.08 Hz, 3H), 2.53 (s, 3H), 2.54 (s, 3H), 3.59 (q, J = 7.13 Hz, 2H), 5.12 (s, 2H), 6.82 (d, J =
7
.22 Hz, 1H), 6.90 (t, J = 8.24 Hz, 1H), 7.01 (d, J = 8.38 Hz, 1H), 7.06 (d, J = 8.09 Hz, 1H), 7.14
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5
5
5
5
6
(
d, J = 8.38 Hz, 1H), 7.23 (dd, J = 10.69, 2.02 Hz, 1H), 8.40 (d, J = 6.94 Hz, 1H). LC−MS m/z
+
4
61 [M + H] , t = 2.89 min.
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8-Chloro-3-cyclopropylmethyl-7-[3-fluoro-4-(isopropylamino)-phenyl][1,2,4]triazolo[4,3-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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2
3
4
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6
7
8
9
0
a]-pyridine (24). Starting from 50a (0.350 g, 1.049 mmol) and 52g (0.381 g, 1.36 mmol) and
following the procedure described for 8, compound 24 was obtained as a white solid (0.097 g,
1
2
6%). Mp 196.4ºC. H NMR (500 MHz, CDCl ) δ ppm 0.35 (q, J = 5.20 Hz, 2H), 0.60 ꢀ 0.67
3
(m, 2H), 1.15 ꢀ 1.26 (m, 1H), 1.29 (d, J = 6.36 Hz, 6H), 3.10 (d, J = 6.65 Hz, H), 3.72 (dq, J =
13.19, 6.49 Hz, 1H), 3.98 (br d, J = 5.20 Hz, 1H), 6.70 ꢀ 6.81 (m, 1H), 6.87 (d, J = 6.94 Hz, 1H),
+
7
.17 ꢀ 7.31 (m, 2H) 7.91 (d, J = 7.22 Hz, 1H). LC−MS m/z 359 [M + H] , t = 3.11 min.
R
8-Trifluoromethyl-3-cyclopropylmethyl-7-[3-fluoro-4-(isopropylamino)-
phenyl][1,2,4]triazolo[4,3-a]-pyridine (25). Starting from 49a (0.350 g, 1.27 mmol) and 52g
(0.461 g, 1.65 mmol) and following the procedure described for 8, compound 25 was obtained as
1
a white solid (0.250 g, 50%). Mp 196.9ºC. H NMR (500 MHz, CDCl ) δ ppm 0.28ꢀ0.42 (m,
3
2
H), 0.57 ꢀ 0.71 (m, 2H), 1.12 ꢀ 1.26 (m, 1H), 1.29 (d, J = 6.4 Hz, 6H), 3.12 (d, J = 6.6 Hz, 2H),
3
.64ꢀ3.77 (m, 1H), 3.96 (d, J = 4.9 Hz, 1H), 6.74 (t, J = 8.4 Hz, 1H), 6.80 (d, J = 7.2 Hz, 1H),
+
7.02 (d, J = 10.1 Hz, 2H), 8.04 (d, J = 6.9 Hz, 1H). LC−MS m/z 393 [M + H] , t = 3.33 min.
R
8-Trifluoromethyl-3-cyclopropylmethyl-7-[3-chloro-4-(isopropylamino)-
phenyl][1,2,4]triazolo[4,3-a]-pyridine (26). Starting from 49a (0.350 g, 1.27 mmol) and 52h
(0.488 g, 1.65 mmol) and following the procedure described for 8, compound 26 was obtained as
1
a white solid (0.250 g, 50%). Mp 230ºC. H NMR (500 MHz, CDCl ) δ ppm 0.29 ꢀ 0.42 (m, 2H),
3
0
.55 ꢀ 0.71 (m, 2H), 1.11 ꢀ 1.27 (m, 1H), 1.31 (d, J = 6.36 Hz, 6H), 3.13 (d, J = 6.65 Hz, 2H),
.73 (dq, J = 13.11, 6.51 Hz, 1H), 4.38 (br d, J = 7.51 Hz, 1H), 6.71 (d, J = 8.67 Hz, 1H), 6.79
3
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Page 29 of 70
Journal of Medicinal Chemistry
1
2
3
4
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6
7
8
9
(
d, J = 7.22 Hz, 1H), 7.16 (dd, J = 8.38, 1.44 Hz, 1H), 7.30 (d, J = 2.02 Hz, 1H), 8.03 (d, J =
+
6
.94 Hz, 1H). LC−MS m/z 409 [M + H] , t = 3.71 min.
R
8-Chloro-3-cyclopropylmethyl-7-[3-fluoro-4-(cyclopropylamino)-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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48
49
50
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56
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60
phenyl][1,2,4]triazolo[4,3-a]-pyridine (27). Starting from 50a (0.30 g, 0.9 mmol) and 52i (0.3
g, 1.08 mmol) and following the procedure described for 8, compound 27 was obtained as a
1
white solid (0.147 g, 46%). H NMR (500 MHz, CDCl ) δ ppm 0.28 ꢀ 0.40 (m, 2H), 0.57 ꢀ 0.69
3
(m, 4H), 0.78 ꢀ 0.87 (m, 2H), 1.15 ꢀ 1.28 (m, 1H), 2.45 ꢀ 2.56 (m, 1H), 3.11 (d, J = 6.65 Hz, 2H),
4.60 (br s, 1H), 6.88 (d, J = 7.22 Hz, 1H), 7.13 ꢀ 7.20 (m, 1H), 7.21 ꢀ 7.31 (m, 2H), 7.92 (d, J =
+
6
.94 Hz, 1H). LC−MS m/z 357 [M + H] , t = 3.30 min.
R
8-Trifluoromethyl-3-cyclopropylmethyl-7-[3-fluoro-4-(cyclopropylamino)-
phenyl][1,2,4]triazolo[4,3-a]-pyridine (28). Starting from 49a (0.20 g, 0.73 mmol) and 52i
(0.221 g, 0.8 mmol) and following the procedure described for 8, compound 28 was obtained as
1
a white solid (0.169 g, 60%). Mp. > 300ºC. H NMR (400 MHz, CDCl ) δ ppm 0.28 ꢀ 0.43 (m,
3
2H), 0.51 ꢀ 0.72 (m, 4H), 0.75 ꢀ 0.91 (m, 2H), 1.12 ꢀ 1.31 (m, 1H), 2.50 (tt, J = 6.56, 3.27 Hz,
1H), 3.13 (d, J = 6.70 Hz, 2H), 4.58 (br s, 1H), 6.80 (d, J = 6.94 Hz, 1H), 6.98 ꢀ 7.09 (m, 2H),
+
7.10 ꢀ 7.18 (m, 1H), 8.05 (d, J = 7.17 Hz, 1H). LC−MS m/z 391 [M + H] , t = 3.18 min.
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8-Trifluoromethyl-3-cyclopropylmethyl-7-[3-chloro-4-(cyclopropylamino)-
phenyl][1,2,4]triazolo[4,3-a]-pyridine (29). Starting from 49a (0.20 g, 0.73 mmol) and 52j
(0.34 g, 0.8 mmol) and following the procedure described for 8, compound 29 was obtained as a
1
white solid (0.109 g, 37%). H NMR (400 MHz, CDCl ) δ ppm 0.31 ꢀ 0.40 (m, 2H), 0.59 ꢀ 0.69
3
(m, 4H), 0.81 ꢀ 0.89 (m, 2H), 1.15 ꢀ 1.27 (m, 1H), 2.46 ꢀ 2.56 (m, 1H), 3.13 (d, J = 6.70 Hz, 2H),
4.93 (s, 1H), 6.80 (d, J = 7.17 Hz, 1H), 7.11 ꢀ 7.16 (m, 1H), 7.17 ꢀ 7.23 (m, 1H), 7.28 (d, J = 1.85
+
Hz, 1H), 8.05 (d, J = 7.17 Hz, 1H). LC−MS m/z 407 [M + H] , t = 3.48 min.
R
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